1. Field of the Invention
The present invention relates to minimizing the interfering effects of certain reducing agents on the analysis of a component in a liquid test solution containing one or more of these interfering substances.
The art of analytical chemistry has been greatly advanced since biochemistry began emerging as a primary scientific frontier requiring increasingly sophisticated analytical methods and tools to solve problems, the solutions of which were never before attempted. Likewise, the medical profession has lent impetus to the growth of analytical chemistry, requiring both high precision and speed in obtaining results. This remarkable progress has been still further spurred by industries such as brewing, chemical manufacturing, and others.
To satisfy the needs of these expanding technologies, a myriad of analytical procedures, compositions and apparatuses have evolved, including solution chemistry techniques, automated machinery and the so-called "dip-and-read" reagent strips. It is to the last of these that the present invention is primarily directed, although substantial benefit ultimately inures to the other procedures as well.
Reagent strip type test devices enjoy wide use in many analytical applications, especially in the chemical analysis of biological fluids, because of their relative low cost, ease of utilizability and speed in obtaining results. In medicine, for example, numerous physiological functions can be monitored merely by dipping reagent strips into a sample of body fluid, such as urine, and observing a detectable response such as a change in color or a change in the amount of light reflected from or absorbed by the strip.
Compatible with such "dip-and-read" methods have arisen many chemistries for detecting body fluid components. Most of these produce a detectable response which is quantitative or at least semi-quantitative. Thus, by measuring the response after a predetermined time, the analyst can obtain not only a positive indication of the presence of a particular constituent in a test sample, but also an estimate of how much of the constituent is present. Such strips provide the physician with a facile diagnostic tool as well as the ability to gauge the extent of disease or bodily malfunction.
Illustrative of dip-and-read strips currently in use are products available from the Ames Division of Miles Laboratories, Inc. under the trademarks CLINISTIX.RTM., MULTISTIX.RTM., KETOSTIX.RTM., N-MULTISTIX.RTM.-C, DIASTIX.RTM., DEXTROSTIX.RTM., and others. Test devices such as these usually comprise one or more carrier matrices, such as absorbent paper, having respectively incorporated with them a particular reactant system which manifests a color change in the presence of a specific test sample components. Depending on the reactant system incorporated with a particular matrix, these devices can detect the presence of glucose, ketone bodies, bilirubin, occult blood, nitrite, and other pathological substances. The specific color change and the intensity of the color observable within a specific time range after contacting the strip with the sample is indicative of the presence of a particular component and its concentration in the sample. Some of these test devices and their reactant systems are set forth in U.S. Pat. Nos. 3,123,443 (CLINISTIX.RTM.); 3,212,855 and 4,147,514 (KETOSTIX.RTM.); 3,814,668, 3,164,534 and 2,981,606 (DIASTIX.RTM.); and 3,298,789, 3,092,465, 3,164,534 and 2,981,606 (DEXTROSTIX.RTM.).
2. Description of the Prior Art
The history of sugar analysis is perhaps most noteworthy because it has seen dramatic change over the years, both in the basic chemistries utilized and in its format. For the most part these analyses can be characterized as oxidizing systems which, when reduced, initiate reaction conditions leading to a detectable response, such as a color change or change in wavelength of ultraviolet light absorbed or reflected by the system. Thus, reducing sugars will convert silver oxide to metallic silver, and, if a solution of the sugar is applied to a piece of filter paper impregnated with silver oxide, a black dot develops. F. Feigl, Chem. Ind., Vol. 57, p. 1161, London (1938). Similarly, o-dinitrobenzene and the 3,4-and 3,5-isomers of dinitrophthalic acid give a sensitive color reaction (forming violet shades) when heated with reducing sugars in Na.sub.2 CO.sub.3. T. Momose, et al., Chem. Pharm. Bull. Tokyo, Vol. 12, p. 14 (1964); F. Feigl, Spot Tests in Organic Analysis, 7th Edition, pp. 338-339, Elsevier Publ. Co., New York (1966).
But as early as 1849 it was known that reducing sugars would cause an alkaline solution of CuSO.sub.4 to precipitate the yellow to red Cooper(I)oxide (or oxyhydrate). H. Fehling, Ann., Vol. 72 (1849). See also B. Herstein, J. Am. Chem. Soc., Vol. 32, p. 779 (1910). This early milestone, known as the Fehling test, lent impetus to the development of a far more sensitive test which utilized silver oxide in ammonia, the so-called Tollens reagent, which reacts readily with reducing agents to produce a black precipitate of metallic silver, often forming a mirror on the inside walls of glass reaction vesssels. B. Tollens, Ber., Vol. 14, p. 1950 (1881); Vol. 15, p. 1635, 1828 (1882).
Because of the relatively high incidence of diabetes mellitus and its accompanying serious clinical consequences, high interest from the biological and medical professions arose in new technique for analyzing glucose levels in urine and serum. This keen interest led to the development of several procedures which deviate dramatically from their solution chemistry forbears. These utilize sophisticated biochemical systems which can be incorporated into dry, dip-and-read devices, used in solutions or suspension techniques, or in conjunction with spectrophotometers and other hardware.
Of these new techniques, the present invention lends itself especially well to an enzymatic system wherein the analyte, for instance glucose, is a substrate for a particular enzyme, the reaction products being capable of eliciting a detectable response for chromogenic indicator compounds, such as those known loosely in the art as "benzidine-type indicators". These will be more carefully defined, infra, but for the present suffice it to say these compounds can undergo color changes in the presence of hydrogen peroxide and a peroxidative substance, such as the enzyme peroxidase. The glucose/glucose oxidase system exemplifies the prior art, wherein glucose is oxidized to gluconic acid with the concomitant formation of H.sub.2 O.sub.2 in accordance with: ##STR1##
It is the formation of hydrogen peroxide which facilitates the subsequent, indicator-related steps leading to observable color formation or other detectable response. Thus a benzidine-type indicator responds in the presence of hydrogen peroxide and peroxidase by changing its light absorptive capability.
In practice, this technology is presently utilized for glucose analysis in the form of dip-and-read reagent strips such as those marketed by the Ames Division of Miles Laboratories, Inc. under the trademark CLINISTIX.RTM. and others. Broadly, these comprise a plastic strip, at one end of which is mounted an absorbent paper portion impregnated with the appropriate enzymes, indicator compound and buffering agents as the principal active ingredients. They are used by dipping the reagent-bearing end into the test sample, removing it and comparing any color formed in the paper with a standard color chart calibrated to various glucose concentrations.
Despite the remarkable gains provided by the reagent strips, certain substances often present in the test sample are often found to interfere with the accuracy of the test. When the concentrations of such substances reach a certain threshold level, in comparison to that of the substrate measured, the adverse effect on the test can become marked. For example, those skilled in the art of reagent strips have for a long time been aware that the presence of ascorbic acid in urine can adversely affect the analysis of such nonrelated components as glucose, occult blood, bilirubin, and nitrite. Thus, high urinary concentration of ascorbic acid from therapeutic doses of vitamin C or parenteral preparations which contain vitamin C as a reducing agent; e.g., tetracyclines, can inhibit the reaction of such tests and limit their accuracy.
The prevalence of this long unvolved problem is indeed evidenced by the many attempts to surmount it recorded in the prior art. U.S. Pat. No. 3,411,887 to Ku, and assigned to the present assignee, discloses the use of a metal ion having an oxidation-reduction potential above that of the interfering substance, but below that of the chromogenic substance or indicator. While this approach appears theoretically feasible, and does somewhat minimize interference from ascorbic acid, it reduces the quantitative accuracy of the indicators in sugar-sensitive reagent systems, especially glucose systems. Primarily, the difficulty appears to stem from a severe lack of storage stability of a sugar-sensitive indicator composition in the presence of the metal ion. Because of the reduced shelf life and lack of quantitative response, the compounds described in U.S. Pat. No. 3,411,887 have never performed sufficiently well to produce a reliable sugar test; the cure being worse than the malady.
U.S. Pat. Nos. 3,975,398 and 3,988,208 disclose indicator compounds purported to be uninhibited by acetoacetic acid or ascorbic acid. Other attempts at solving this problem of interference were through the use of ion-exchange substances (British Pat. No. 1,193,594) and multi-layered carrier matrices (British Pat. No. 1,171,788) in hopes that the interfering substances could be physically separated from the test sample prior to contacting the reagent system.
Despite long continued efforts such as those described above, to date no substantial solution to the problem has been devised. The "trapping system" of U.S. Pat. No. 3,411,887, as will be demonstrated by the Examples, infra, provides compositions of marginal stability. Test devices utilizing such technology are virtually useless after just a brief period of storage at room temperature.
The present invention solves this long-felt need for an indicator system having a substantial resistance to interference from reducing agents such as ascorbic acid (vitamin C).